The termination of protein synthesis in ribosomes is governed by termination (stop) codons in messenger RNAs and by polypeptide chain release factors (RFs). Although the primary structure of prokaryotic RFs and yeast mitochrondrial RF is established, that of the only known eukaryotic RF (eRF) remains obscure. Here we report the assignment of a family of tightly related proteins (designated eRF1) from lower and higher eukaryotes which are structurally and functionally similar to rabbit eRF. Two of these proteins, one from human and the other from Xenopus laevis, have been expressed in yeast and Escherichia coli, respectively, purified and shown to be active in the in vitro RF assay. The other protein of this family, sup45 (sup1) of Saccharomyces cerevisiae, is involved in omnipotent suppression during translation. The amino-acid sequence of the eRF1 family is highly conserved. We conclude that the eRF1 proteins are directly implicated in the termination of translation in eukaryotes.